Bioluminescence in G Protein-Coupled Receptors Drug Screening Using Nanoluciferase and Halo-Tag Technology

Schihada, Hannes; Nemec, Katarina; Maiellaro, Isabella

doi:10.1007/978-1-0716-1221-7_9

Cited by 7 publications

(6 citation statements)

References 14 publications

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“…Subsequently, the average ΔRET of buffer-treated control wells was subtracted. To reduce the fluctuation of the BRET ratio, three consecutive BRET ratios were averaged before and after ligand addition ( 65 ). Concentration–response curve experiments were fitted using a three- or four-parameter logistic curve fit as stated in corresponding figure legends.…”

Section: Methodsmentioning

confidence: 99%

Functional modulation of PTH1R activation and signaling by RAMP2

Nemec

Schihada

Kleinau

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Receptor-activity-modifying proteins (RAMPs) are ubiquitously expressed membrane proteins that associate with different G protein–coupled receptors (GPCRs), including the parathyroid hormone 1 receptor (PTH1R), a class B GPCR and an important modulator of mineral ion homeostasis and bone metabolism. However, it is unknown whether and how RAMP proteins may affect PTH1R function. Using different optical biosensors to measure the activation of PTH1R and its downstream signaling, we describe here that RAMP2 acts as a specific allosteric modulator of PTH1R, shifting PTH1R to a unique preactivated state that permits faster activation in a ligand-specific manner. Moreover, RAMP2 modulates PTH1R downstream signaling in an agonist-dependent manner, most notably increasing the PTH-mediated Gi3 signaling sensitivity. Additionally, RAMP2 increases both PTH- and PTHrP-triggered β-arrestin2 recruitment to PTH1R. Employing homology modeling, we describe the putative structural molecular basis underlying our functional findings. These data uncover a critical role of RAMPs in the activation and signaling of a GPCR that may provide a new venue for highly specific modulation of GPCR function and advanced drug design.

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Section: Methodsmentioning

confidence: 99%

Functional modulation of PTH1R activation and signaling by RAMP2

Nemec

Schihada

Kleinau

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…To quantify ligand-induced FRET changes, ΔFRET was calculated for each well and time point as percent over basal ([(FRETstim− FRETbasal)/FRETbasal] × 100). Subsequently, the average ΔFRET of buffer-treated control wells was subtracted (Schihada et al, 2021). Concentration−effect curves were fitted by a three-parameter logistic function yielding parameter values for a ligand's potency (pEC50).…”

Section: Forskolin-induced Pka Phosphorylationmentioning

confidence: 99%

Receptor-associated independent cAMP nanodomains mediate spatiotemporal specificity of GPCR signaling

Anton

Kayser

Maiellaro

et al. 2022

Cell

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117

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“…The FRET-based H 3 R sensor in intact cells and cultured on cover slips allowed for the rapid detection of ligand-induced conformational receptor changes using a fluorescent microscope equipped with a perfusion system with high temporal resolution but a relatively low throughput [ 22 ]. Substitution of YFP and CFP with respectively a red fluorescent dye covalently bound to HaloTag and NanoLuc allows a NanoBRET-based detection of conformational H 3 R changes in living cells using a 96-well plate reader-based format to readily generate full concentration-response curves for multiple ligands in a single assay run [ 12 ], as also previously optimized and reported for the α 2A -adrenergic receptor, β 2 -adrenergic receptor, and the parathyroid hormone 1 receptor [ 11 , 21 ]. As a follow-up on our initial report on this sensor, we evaluated a number of well-known H 3 R tools (photo-switchable ligands or preclinical candidates) for their conformational effects.…”

Section: Discussionmentioning

confidence: 99%

“…Initially, cyan and yellow fluorescent proteins (CFP and YFP) were used as fluorescence resonance energy transfer (FRET) donor and acceptor, respectively, in intramolecular GPCR conformation sensors to measure the distance/re-orientation between IL3 and the C-terminal tail, with the substitution of YFP with the much smaller Fluorescein Arsenical Hairpin Binder (FlAsH) as an improved alternative due to its reduced effect on the GPCR structure [ 14 , 15 , 16 ]. More recently, bioluminescent luciferases such as Renilla luciferase or the engineered NanoLuc in combination with fluorescent proteins, FlAsH, or the self-labeling fluorescent HaloTag have been employed in BRET-based GPCR conformation sensors to allow for the measurements of conformational changes in GPCRs in a microplate reader assay format [ 11 , 12 , 17 , 18 , 19 , 20 , 21 ].…”

Section: Introductionmentioning

confidence: 99%

A NanoBRET-Based H3R Conformational Biosensor to Study Real-Time H3 Receptor Pharmacology in Cell Membranes and Living Cells

Gao

Vischer

et al. 2022

IJMS

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Conformational biosensors to monitor the activation state of G protein-coupled receptors are a useful addition to the molecular pharmacology assay toolbox to characterize ligand efficacy at the level of receptor proteins instead of downstream signaling. We recently reported the initial characterization of a NanoBRET-based conformational histamine H3 receptor (H3R) biosensor that allowed the detection of both (partial) agonism and inverse agonism on living cells in a microplate reader assay format upon stimulation with H3R ligands. In the current study, we have further characterized this H3R biosensor on intact cells by monitoring the effect of consecutive ligand injections in time and evaluating its compatibility with photopharmacological ligands that contain a light-sensitive azobenzene moiety for photo-switching. In addition, we have validated the H3R biosensor in membrane preparations and found that observed potency values better correlated with binding affinity values that were measured in radioligand competition binding assays on membranes. Hence, the H3R conformational biosensor in membranes might be a ready-to-use, high-throughput alternative for radioligand binding assays that in addition can also detect ligand efficacies with comparable values as the intact cell assay.

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Bioluminescence in G Protein-Coupled Receptors Drug Screening Using Nanoluciferase and Halo-Tag Technology

Cited by 7 publications

References 14 publications

Functional modulation of PTH1R activation and signaling by RAMP2

Functional modulation of PTH1R activation and signaling by RAMP2

Receptor-associated independent cAMP nanodomains mediate spatiotemporal specificity of GPCR signaling

A NanoBRET-Based H3R Conformational Biosensor to Study Real-Time H3 Receptor Pharmacology in Cell Membranes and Living Cells

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